Midkine (hereinafter, referred to as “MK”) is a growth/differentiation factor found as a product of a gene transiently expressed in the stage of retinoic acid-induced differentiation of embryonal carcinoma (EC) cells and is a polypeptide of 13 kDa in molecular weight rich in basic amino acids and cysteine (see e.g., Non-Patent Documents 1 and 2).
MK is known to have various biological activities. It is known that MK expression is increased in human cancer cells. This increase in expression has been confirmed in various cancers such as esophageal cancer, thyroid cancer, urinary bladder cancer, colon cancer, stomach cancer, pancreatic cancer, thoracic cancer, liver cancer, lung cancer, breast cancer, neuroblastoma, glioblastoma, uterine cancer, ovarian cancer, and Wilms tumor (see e.g., Patent Document 1 and Non-Patent Document 3). Moreover, MK is thought to promote the survival and migration of cancer cells, promote angiogenesis, and contribute to cancer progression.
Moreover, MK is known as a molecule that plays a central role in the stage of inflammation formation. For example, it is known that neointimal formation after vascular injury and nephritis onset during ischemic injury are suppressed in knockout mice deficient in MK genes. Moreover, it is also known that rheumatism models and postoperative adhesion are significantly suppressed in such knockout mice (see e.g., Patent Documents 2, 3, and 4). Thus, MK is known to participate in inflammatory diseases such as arthritis, autoimmune disease, rheumatic arthritis (rheumatoid arthritis (RA) or osteoarthritis (OA)), multiple sclerosis, postoperative adhesion, inflammatory bowel disease, psoriasis, lupus, asthma, and neutrophil dysfunction. Furthermore, MK is known to promote the movement (migration) of inflammatory cells such as macrophages or neutrophils. Since this movement is necessary for the establishment of inflammation, it is thought that deficiency of MK probably prevents diseases based on inflammation (see e.g., Patent Document 5).
Moreover, since MK exists at an increased level in peritoneal fluids of women with progressive endometriosis and stimulates the growth of cultured endometrial stromal cells, MK is known to participate in the onset and progression of endometriosis (see e.g., Patent Document 6).
Furthermore, since MK has vascular intimal thickening effects, MK is known to participate in occlusive vascular diseases such as post-revascularization restenosis, coronary occlusive disease, cerebrovascular occlusive disease, renovascular occlusive disease, peripheral occlusive disease, arteriosclerosis, and cerebral infarction (see e.g., Patent Document 2).
Cell migration is known to be important for mechanisms underlying cancer cell infiltration/metastasis, intimal thickening in atherosclerotic lesions, angiogenesis, and so on. Moreover, inflammatory cell migration is known to deeply participate in cardiovascular diseases such as angina pectoris, myocardial infarction, cerebral infarction, cerebral hemorrhage, and hypertension.
Moreover, the three-dimensional structure of MK has been determined by NMR and reported (see e.g., Non-Patent Document 4). MK is composed of: an N-terminal fragment (hereinafter, referred to as an “N-fragment”) consisting of amino acid residues 1 to 52; a C-terminal fragment (hereinafter, referred to as a “C-fragment”) consisting of amino acid residues 62 to 121; and a loop region (amino acid residues 53 to 61) (hereinafter, referred to as a “loop”) that links these fragments.
Each of the N- and C-fragments is mainly composed of: a portion having a three-dimensional structure consisting of three antiparallel β-sheets (hereinafter, referred to as a “domain”; the domain (consisting of amino acid residues 15 to 52) in the N-fragment is referred to as an “N-domain”, and the domain (consisting of amino acid residues 62 to 104) in the C-fragment is referred to as a “C-domain”); and a terminally located portion devoid of the domain that does not assume a particular three-dimensional structure (hereinafter, referred to as a “tail”; the tail (consisting of amino acid residues 1 to 14) in the N-fragment is referred to as an “N-tail”, and the tail (consisting of amino acid residues 105 to 121) in the C-fragment is referred to as a “C-tail”). Basic amino acids on the C-domain surface form two clusters: a cluster consisting of lysine 79, arginine 81, and lysine 102 (cluster I) and a cluster consisting of lysine 86, lysine 87, and arginine 89 (cluster II) (see e.g., Non-Patent Document 4). Both the clusters are known to participate in heparin-binding ability (see e.g., Non-Patent Documents 4 and 5).
On the other hand, regarding antibodies against MK, mice immunized with human MK proteins as immunogens are considered to hardly recognize the MK proteins as foreign heterologous proteins in vivo, due to high conservation of MK proteins among species and 87% homology between human and mouse MK amino acid sequences. Thus, it is known that anti-human MK monoclonal antibodies are obtained using MK gene-knockout mice (see e.g., Patent Document 7). However, there have been no previous reports on epitopes on MK recognized by anti-human MK antibodies having the activity of inhibiting MK functions.    Patent Document 1: Japanese Patent Laid-Open No. 6-172218    Patent Document 2: Pamphlet of WO2000/10608    Patent Document 3: Pamphlet of WO2004/078210    Patent Document 4: Pamphlet of WO2004/085642    Patent Document 5: Pamphlet of WO1999/03493    Patent Document 6: Pamphlet of WO2006/016571    Patent Document 7: Japanese Patent Laid-Open No. 2002-85058    Non-Patent Document 1: Kadomatsu, K. et al.: (1988) Biochem. Biophys. Res. Commun., 151: p. 1312-1318    Non-Patent Document 2: Tomokura, M. et al.: (1999) J. Biol. Chem, 265: p. 10765-10770    Non-Patent Document 3: Muramatsu, T.: (2002) J. Biochem. 132, p. 359-371    Non-Patent Document 4: Iwasaki, W. et al.: (1997) EMBO J. 16, p. 6936-6946    Non-Patent Document 5: Asai, S. et al.: (1995) Biochem. Biophys. Res Commun. 206, P. 468-473.